Construction of chimeric antigen receptor targeting CD20 antigen and activity identification of engineered T cells thereof

ABSTRACT

Provided are a chimeric antigen receptor targeting CD20 antigen and a preparation method thereof. The extracellular antigen binding domain of the chimeric antigen receptor includes an antibody heavy chain variable region shown in SEQ ID NO: 7 or 9 or 33 and an antibody light chain variable region shown in SEQ ID NO: 11 or 13 or 35, and is capable of killing tumor cells.

TECHNICAL FIELD

The present invention provides a sequence component of chimeric antigenreceptor targeting CD20 antigen, and a preparation method for itsmodified T cells (CART20) and activity identification thereof. Thepresent invention identifies a chimeric antigen receptor structure fortreating CD20 positive B cell lymphoma.

BACKGROUND TECHNIQUE

Malignant tumors of the blood system account for about 10% of humanmalignant tumors, and 95% of malignant tumors of the blood system arederived from B lymphocytes. Traditional chemotherapy and radiotherapyplay an important role in the treatment of malignant tumors of the bloodsystem. Some patients also have significant effects, but most of themare difficult to cure. New and effective treatments have been a hottopic in this field.

Adoptive T cell therapy has shown its powerful efficacy and brightprospect in the clinical treatment of malignant tumors. Among them,multiple centers independently using Chimeric Antigen Receptor(CAR)-modified T cells to target recurrent, refractory malignant tumorsof CD19-expressed B cell have achieved unprecedented success. Inparticular, in a clinical trial carried out at the School of Medicine,University of Pennsylvania using CART19 in the treatment of recurrent,refractory acute B-cell lymphoma (R/R B-ALL), up to 94% of patientsachieved complete remission. Although the initial response rate of thisclinical trial was high, nearly 40% of patients who achieved completeresponse after 1 month of treatment, had a relapse, and more than 60% ofpatients with relapse had CD19-negative tumor cells escape. Therefore,there is an urgent need to screen out CART structure that target B celllymphoma-associated antigens other than CD19 to treat patients withmalignant lymphoma.

CD20 is a glycosylated protein and is the first identified B cellmembrane marker. CD20 is also known as B 1, and encoded by the MS4Agene. CD20 molecule has four transmembrane hydrophobic regions, and itsN-terminal and C-terminal are located on the cytoplasmic side, therebyforming two closed loops outside the cell, which are respectively calledbig loop and small loop. CD20 is specifically expressed in more than 95%of normal and cancerous B cells. These cells are in the pre-B cell stageand subsequent developmental stages, and CD20 stops expression until thecells differentiated into plasma cells. Therefore, CD20 is an idealtarget for immunotherapy of B cell malignancies.

Rituximab (MabThera®, Rituxan®) is the first generation of chimericmonoclonal antibody targeting CD20 which is firstly approved by the USFDA and the European EMA for treating indolent lymphoma. Rituximabrecognizes and binds to the big loop structure of the extracellulardomain of CD20, and it kills tumor cells by ADCC-mediated killingeffect. However, Rituximab alone shows limited activity and shortduration of response, but its combination with chemotherapy cansignificantly enhance the efficacy of chemotherapy. Rituximab is usedfor the treatment of lymphoma, and half of the patients have a completeresponse (CR) or a partial response (PR).

Ofatumumab (Arzerra®) is the first completely humanized CD20 therapeuticantibody. Unlike Rituximab, the epitope recognized by Ofatumumabcontains parts of the big loop and the small loop of CD20. At the sametime, the tumor killing method of Ofatumumab is mainly through thecomplement-dependent pathway, followed by ADCC-dependent tumor killingeffect.

Obinutuzumab (Gazyvaro®, Gazyva®) is a humanized type II CD20 antibodythat reduces fucosylation levels and optimizes FcγRIIIa affinity.Obinutuzumab recognizes and binds to the big loop of the extracellularmolecule of CD20, and mediates the killing effect on tumor mainlythrough the ADCC effect. At the same time, the binding of Obinutuzumabto CD20 molecule also has the effect of inducing apoptosis of tumorcells. As for the NHL that does not respond to Rituximab treatment,Obinutuzumab is combined with bendamustine, a nitrogen mustard drug. Thephase III clinical trial found that the duration with no deteriorationof combination therapy of Obinutuzumab and bendamustine was twice aslong as that of bendamustine therapy alone (the former is 29 months andthe latter is 14 months). Obinutuzumab has an overall response rate(ORR, including CR and PR) of 77.3%, and Rituximab is 65.7%.

Compared with therapeutic antibodies, cellular immunotherapy is anemerging and highly effective tumor treatment model, and is a new typeof autoimmunolgy treatment for cancer. It is a method for in vitroculture and amplification of immune cells collected from a patient usingbiotechnology and biological agents, and then the cells are transfusedback to the patient to stimulate and enhance the body's autoimmunefunction, thereby achieving the purpose of treating tumors. The skilledin the art have been working to develop new cellular immunotherapy toincrease its efficiency and reduce its side effect. Although manytherapeutic antibodies as described above have been developed in theseyears, their clinical therapeutic effects have not reached the samelevel of therapeutic effects as CART19. Therefore, the development ofCART therapy targeting CD20 has great market value and socialsignificance.

SUMMARY OF THE INVENTION

In view of the differences in affinity and killing mechanisms of thetherapeutic antibodies targeting CD20, we constructed a series ofchimeric antigen receptors targeting CD20 using the DNA sequences of theantigen-binding regions of different antibodies, and completed theidentification of anti-tumor activity and differential comparison ofthese chimeric antigen receptor engineering T cells in vitro. Theinvention provides new and effective methods and preparations forclinical application of CAR-T in the treatment of CD20-positive leukemiaand lymphoma.

It is an object of the present invention to provide a chimeric antigenreceptor targeting CD20, a preparation method and application thereof.

The present invention relates to the construction of a chimeric antigenreceptor structure targeting CD20, a preparation method of a chimericantigen receptor engineered T cell targeting CD20, and activityidentification thereof.

In a first aspect of the invention, it provides a chimeric antigenreceptor (CAR) (sequence), whose antigen binding domain (i.e., scFv)comprises an antibody heavy chain variable region as shown in SEQ IDNOs: 7 or 9 or 33 and an antibody light chain variable region as shownin SEQ ID NOs: 11 or 13 or 35.

In another preferred embodiment, the antigen binding domain of thechimeric antigen receptor is as follows:V_(H)-V_(L)

wherein V_(H) is an antibody heavy chain variable region; V_(L) is anantibody light chain variable region; and “-” is a linker peptide or apeptide bond.

In another preferred embodiment, the amino acid sequence of the linkerpeptide is as shown in SEQ ID NO: 15.

In another preferred embodiment, the amino acid sequence of V_(H) is asshown in SEQ ID NO: 7, and the amino acid sequence of V_(L) is as shownin SEQ ID NO: 11.

In another preferred embodiment, the amino acid sequence of V_(H) is asshown in SEQ ID NO: 9, and the amino acid sequence of V_(L) is as shownin SEQ ID NO: 13.

In another preferred embodiment, the amino acid sequence of V_(H) is asshown in SEQ ID NO: 33, and the amino acid sequence of V_(L) is shown inSEQ ID NO: 35.

In another preferred embodiment, the structure of the chimeric antigenreceptor is as follows:L-V_(H)-V_(L)-H-TM-CS-CD3ζ

wherein,

L is an optional leader sequence (i.e., signal peptide sequence);

H is a hinge region;

TM is a transmembrane domain;

CS is a co-stimulatory molecule derived from 4-1BB and/or CD28;

CD3ζ is a cytoplasmic signaling sequence derived from CD3ζ;

V_(H), V_(L), and “-” are as described above, respectively.

In another preferred embodiment, the sequence of L is as shown in SEQ IDNO: 27.

In another preferred embodiment, the sequence of H is as shown in SEQ IDNO: 17 or 19.

In another preferred embodiment, the sequence of TM comprises atransmembrane region derived from CD8a or CD28, preferably the sequenceof TM is as shown in SEQ ID NO: 21 or 37.

In another preferred embodiment, the CS structure is: CD28-4-1BB,wherein CD28 is a co-stimulatory molecule derived from CD28; and 4-1BBis a co-stimulatory molecule derived from 4-1BB.

In another preferred embodiment, the sequence of the co-stimulatorymolecule derived from 4-1 BB is as shown in SEQ ID NO: 23.

In another preferred embodiment, the sequence of the co-stimulatorymolecule derived from CD28 is as shown in SEQ ID NO: 39.

In another preferred embodiment, the sequence of CD3ζ is as shown in SEQID NO: 25.

In another preferred embodiment, the sequence of the chimeric antigenreceptor is as shown in SEQ ID NOs: 1, 3, 5, 29, or 31.

In a second aspect of the invention, a nucleic acid molecule isprovided, encoding the chimeric antigen receptor (CAR) of the firstaspect of the invention.

In another preferred embodiment, the nucleic acid molecule comprises anucleic acid sequence encoding the hinge region selected from the groupconsisting of:

(a) a polynucleotide encoding a polypeptide as shown in SEQ ID NO: 17 or19;

(b) a polynucleotide having a sequence as shown in SEQ ID NO: 18 or 20;

(c) a polynucleotide having a nucleotide sequence with >90%(preferably >95%) homologous to the sequence of SEQ ID NO: 18 or 20, andencoding the amino acid sequence of SEQ ID NO: 17 or 19;

(d) a polynucleotide complementary to the polynucleotide of any of (a)to (c).

In another preferred embodiment, the nucleic acid molecule comprises anucleic acid sequence encoding the CD8a transmembrane region selectedfrom the group consisting of:

(a) a polynucleotide encoding a polypeptide as shown in SEQ ID NO: 21;

(b) a polynucleotide having a sequence as shown in SEQ ID NO: 22;

(c) a polynucleotide having a nucleotide sequence with >90%(preferably >95%) homologous to the sequence of SEQ ID NO: 22 andencoding the amino acid sequence of SEQ ID NO: 21;

(d) a polynucleotide complementary to the polynucleotide of any of (a)to (c).

In another preferred embodiment, the nucleic acid molecule comprises anucleic acid sequence encoding the 4-1BB (CD137) intracellular signaldomain selected from the group consisting of:

(a) a polynucleotide encoding a polypeptide as shown in SEQ ID NO: 23;

(b) a polynucleotide having a sequence as shown in SEQ ID NO: 24;

(c) a polynucleotide having a nucleotide sequence with >90%(preferably >95%) homologous to the sequence of SEQ ID NO: 24 andencoding the amino acid sequence of SEQ ID NO: 23;

(d) a polynucleotide complementary to the polynucleotide of any of (a)to (c).

In another preferred embodiment, the nucleic acid molecule comprises anucleic acid sequence encoding the CD28 intracellular signal domainselected from the group consisting of:

(a) a polynucleotide encoding a polypeptide as shown in SEQ ID NO: 39;

(b) a polynucleotide having a sequence as shown in SEQ ID NO: 40;

(c) a polynucleotide having a nucleotide sequence with >90%(preferably >95%) homologous to the sequence of SEQ ID NO: 40 andencoding the amino acid sequence of SEQ ID NO: 39;

(d) a polynucleotide complementary to the polynucleotide of any of (a)to (c).

In another preferred embodiment, the nucleic acid molecule comprises anucleic acid sequence encoding the CD3 intracellular signal domainselected from the group consisting of:

(a) a polynucleotide encoding a polypeptide as shown in SEQ ID NO: 25;

(b) a polynucleotide having a sequence as shown in SEQ ID NO: 26;

(c) a polynucleotide having a nucleotide sequence with >90%(preferably >95%) homologous to the sequence of SEQ ID NO: 26 andencoding the amino acid sequence of SEQ ID NO: 25;

(d) a polynucleotide complementary to the polynucleotide of any of (a)to (c).

In another preferred embodiment, the nucleic acid molecule comprises anucleic acid sequence selected from the group consisting of:

(a) a polynucleotide encoding a polypeptide as shown in SEQ ID NOs: 1,35, 29 or 31;

(b) a polynucleotide having the sequence as shown in SEQ ID NOs: 2, 4,6, 30 or 32;

(c) a polynucleotide having a nucleotide sequence with >95%(preferably >98%) homologous to the sequence of SEQ ID NOs: 2, 4, 6, 30or 32, and encoding the amino acid sequence of SEQ ID NOs: 1, 35, 29 or31;

(d) a polynucleotide complementary to the polynucleotide of any of (a)to (c).

In another preferred embodiment, the nucleic acid molecule is isolated.

In another preferred embodiment, the nucleic acid molecule furthercomprises a polynucleotide encoding the leader sequence (directingsequence, signal peptide), and the amino acid sequence of the leadersequence is as shown in SEQ ID NO: 27; preferably the polynucleotideencoding the leader sequence (signal peptide) is as shown in SEQ ID NO:28.

In another preferred embodiment, the sequence of the nucleic acidmolecule is as shown in SEQ ID NOs: 2, 46, 30 or 32.

In a third aspect of the invention, it provides a vector, comprising thenucleic acid molecule of the second aspect of the invention.

In another preferred embodiment, the vector is a lentiviral vector.

In a fourth aspect of the invention, it provides a host cell comprisingthe vector of the third aspect of the invention or having the exogenousnucleic acid molecule of the second aspect of the invention integratedinto its genome.

In another preferred embodiment, the cell is an isolated cell, and/orthe cell is a genetically engineered cell.

In another preferred embodiment, the cell is a mammalian cell.

In another preferred embodiment, the cell is a T cell.

In a fifth aspect of the invention, it provides a pharmaceuticalcomposition comprising a pharmaceutically acceptable carrier and thechimeric antigen receptor of the first aspect of the invention, thenucleic acid molecule of the second aspect of the invention, the vectorof the third aspect of the invention, or the cell of the fourth aspectof the invention.

In a sixth aspect of the invention, it provides the use of the chimericantigen receptor of the first aspect of the invention, the nucleic acidmolecule of the second aspect of the invention, the vector of the thirdaspect of the invention, or the cell of the fourth aspect of theinvention for the preparation of a medicine or a formulation fortreating tumor or autoimmune disease.

In another preferred embodiment, the autoimmune disease is an autoimmunedisease caused by overexpression of B cells (such as lupuserythematosus).

In another preferred embodiment, the tumor comprises CD20 positivetumor.

In a seventh aspect of the invention, it provides a method for treatinga disease comprising administering an appropriate amount of the chimericantigen receptor of the first aspect of the invention, the nucleic acidmolecule of the second aspect of the invention, the vector of the thirdaspect of the invention, the cell of the fourth aspect of the invention,or the pharmaceutical composition of the fifth aspect of the invention,to a subject in need of treatment.

In another preferred embodiment, the disease is tumor.

In an eighth aspect of the invention, it provides a method for preparinga CAR-T cell (CAR-modified T cell) expressing the chimeric antigenreceptor of the first aspect of the invention, comprising the steps of:transducing the nucleic acid molecule of the second aspect of theinvention or the vector of the third aspect of the invention into a Tcell, thereby obtaining the CAR-T cell.

It is to be understood that the various technical features of thepresent invention mentioned above and the various technical featuresspecifically described hereinafter (as in the Examples) may be combinedwith each other within the scope of the present invention to constitutea new or preferred technical solution, which will not be repeated one byone herein.

DESCRIPTION OF DRAWINGS

FIG. 1 shows the structure of the chimeric antigen receptor targetingCD20. Each element of the designed CAR structure is shown in the figure,and the listed elements include: a leader sequence, an antigenrecognition sequence (Ofatumumaband, Obinutuzumab, Rituximab), a hingeregion, a transmembrane region, a co-stimulatory factor signal region,and a CD3ζ signaling region. CAR-T20.14, CAR-T20.13 and CAR-T20.16 areCAR structures constructed based on the antibody variable regionsequences of Ofatumumab, Obinutuzumab and Rituxmab, respectively.CAR-T20.19 and CAR-20.20 are the mutant form of CAR-T20.14, havingL235E-N297Q mutation in IgG4 Hinge-CH2-CH3 linker region. CAR-T20.20 isa third generation chimeric antigen receptor structure with codingsequences of both CD28 and 4-1BB co-stimulatory signaling molecule.

FIGS. 2A and 2B show detection of transfection efficiency of engineeredT cell with chimeric antigen receptors targeting CD20. The expressionlevel of the CAR gene-encoded protein on the surface of the T cellmembrane in CAR-T20s cells cultured on day 7 (FIG. 2A) and day 11 (FIG.2B) was identified by the Protein L method.

FIGS. 3A and 3B. 1*10⁵ of NT, CART-20.13, CART-20.14 and CAR-T20.16cells (cultured on day 6) were co-cultured respectively withCD20-positive RAJI and RAMOS tumor cell lines, and CD20-negative MOLT-4tumor cell line in 200 μl GT-551medium for 18 h in a ratio of 1:1. Thenthe expression level of CD137 on the surface of T cell membrane (FIG.3A) and the secretion level of IFNγ in the co-culture supernatant (FIG.3B) were detected.

FIG. 4 shows detection of apoptosis levels of tumor cells induced byCART-20. 1*10⁴ of CFSE-labeled CD20-negative (MOLT-4) or CD20-positive(RAJI, RAMOS) tumor cell lines were co-cultured respectively with NT,CART-20.13, CART-20.14 and CAR-T20.16 cells (cultured on day 11) in 200μl GT-551 medium for 4 h according to the ratio as shown in figure. Thenthe cell pellet was collected by centrifugation. The cells were washedtwice with PBS and stained for 30 min with Annexin V-APC dye in a ratioof 1:50 in 100 μl of dyeing solution. After washing with PBS for 1 time,the proportion of Annexin V positive cells in CFSE positive cells wasanalyzed on a flow cytometry. The results in figure show the statisticalanalysis of Annexin V positive cells in the corresponding co-culturesamples.

FIGS. 5A-5C show identification of the activation ability in vitro ofthe third generation chimeric antigen receptor and the chimeric antigenreceptor with mutation in hinge region (which are constructed based onthe sequence of Ofatumumaband antibody). The expression level of the CARgene-encoded protein (FIG. 5A) on the surface of the T cell membrane inCAR-T20.14, CAR-T20.19 and CAR-T20.20 cells cultured on day 7 wasidentified by the Protein L method. 1*10⁵ of NT, CART-20.14, CART-20.19and CAR-T20.20 cells (cultured on day 7) were cultured respectively withK562, K562 stable transfected cells of CD19 single positive, CD20 singlepositive, CD19 and CD20 double positive, and RAJI target cell in 200 μlGT-551 medium for 18 h in a ratio of 1:1. Then the expression level ofCD137 on the surface of T cell membrane (FIG. 5B) and the secretionlevel of IFNγ in the culture supernatant (FIG. 5C) were detected,respectively.

FIG. 6 shows the detection results of the ability of CAR-T20 cells toscavenge CD20-positive cells in vivo. The results indicate thatCAR-T20.19 can effectively inhibit the in vivo expansion ofCD20-positive tumor cells.

MODES FOR CARRYING OUT THE PRESENT INVENTION

After extensive and intensive studies, the inventors have obtained achimeric antigen receptor targeting CD20 and the preparation andapplication thereof. The extracellular antigen binding domain of thechimeric antigen receptor includes the antibody heavy chain variableregion shown in SEQ ID NO: 1 and the antibody light chain variableregion shown in SEQ ID NO: 2. The experimental results show that thechimeric antigen receptor provided by the present invention showsextremely high killing ability against tumor cells.

In view of the differences in affinity, killing mechanism of therapeuticantibodies targeting CD20, as well as the significant effects ofdifferent transmembrane domains and intracellular domains on theactivity of chimeric antigen receptor, a series of chimeric antigenreceptors targeting CD20 were constructed in the present invention bycombining various transmembrane and intracellular components with theamino acid sequences of the variable regions in various anti-CD20antibodies. The expression of such chimeric antigen receptors in primaryT cells was completed. The detection method of receptor expressionintensity was established. The ability of the CAR-T cells to recognizeCD20 antigen in vitro and in vivo, as well as the difference in theactivity of scavenging malignant tumors carrying CD20 antigen in vitroand in vivo were identified, providing a new effective method andpreparation for the clinical application of CAR T in treating CD20positive leukemia and lymphoma.

Chimeric Antigen Receptor

The invention provides a chimeric antigen receptor (CAR) comprising anextracellular domain, a transmembrane domain, and an intracellulardomain. The extracellular domain comprises a target-specific bindingelement (also known as an antigen binding domain). The intracellulardomain includes a co-stimulatory signaling region and a ζ chain moiety.The co-stimulatory signaling region refers to a part of theintracellular domain that includes a co-stimulatory molecule. Theco-stimulatory molecule is a cell surface molecule required forefficient response of lymphocytes to antigens, rather than an antigenreceptor or its ligand.

A linker can be incorporated between the extracellular domain and thetransmembrane domain of the CAR, or between the cytoplasmic domain andthe transmembrane domain of the CAR. As used herein, the term “linker”generally refers to any oligopeptide or polypeptide that plays a role oflinking the transmembrane domain to the extracellular domain or thecytoplasmic domain in a polypeptide chain. The linker may comprise 0-300amino acids, preferably 2-100 amino acids and most preferably 3-50 aminoacids.

In a preferred embodiment of the invention, the extracellular domain ofthe CAR provided by the invention comprises an antigen binding domaintargeting CD20. When the CAR of the present invention is expressed in Tcell, antigen recognition can be performed based on antigen bindingspecificity. When it binds to its cognate antigen, it affects a tumorcell so that the tumor cell fails to grow, is prompted to die, orotherwise is affected so that the tumor burden in a patient isdiminished or eliminated. The antigen binding domain is preferably fusedwith an intracellular domain from one or more of a co-stimulatorymolecule and a ζ chain. Preferably, the antigen binding domain is fusedwith an intracellular domain of a combination of a 4-1BB signalingdomain and/or a CD28 signaling domain, and a CD3ζ signaling domain.

In one embodiment, the CAR targeting CD20 of the invention comprises thespecific signaling domain of the invention (the transmembrane region ofCD8, the intracellular signal domains of CD137 and CD3ζ are made inseries). The signaling domain of the invention significantly increasesanti-tumor activity and in vivo persistence of CAR-T cells compared toan otherwise identical CAR targeting CD20.

In a preferred embodiment of the invention, the amino acid sequence ofthe chimeric antigen receptor (CAR) provided by the present invention isas follows:

CAR-T20.13 (SEQ ID NO: 29)MALPVTALLL PLALLLHAAR PQVQLVQSGA EVKKPGSSVK VSCKASGYAF SYSWINWVRQ 60APGQGLEWMG RIFPGDGDTD YNGKFKGRVT ITADKSTSTA YMELSSLRSE DTAVYYCARN 120VFDGYWLVYW GQGTLVTVSS GGGGSGGGGS GGGGSDIVMT QTPLSLPVTP GEPASISCRS 180SKSLLHSNGI TYLYWYLQKP GQSPQLLIYQ MSNLVSGVPD RFSGSGSGTD FTLKISRVEA 240EDVGVYYCAQ NLEITYTEGG GTKVEIKRTV ESKYGPPCPP CPAPEFLGGP SVFLFPPKPK 300DTLMISRTPE VTCVVVDVSQ EDPEVQFNWY VDGVEVHNAK TKPREEQFNS TYRVVSVLTV 360LHQDWLNGKE YKCKVSNKGL PSSIEKTISK AKGQPREPQV YTLPPSQEEM TKNQVSLTCL 420VKGFYPSDIA VEWESNGQPE NNYKTTPPVL DSDGSFFLYS RLTVDKSRWQ EGNVFSCSVM 480HEALHNHYTQ KSLSLSLGKI YIWAPLAGTC GVLLLSLVIT LYCKRGRKKL LYIFKQPFMR 540PVQTTQEEDG CSCRFPEEEE GGCELRVKFS RSADAPAYKQ GQNQLYNELN LGRREEYDVL 600DKRRGRDPEM GGKPRRKNPQ EGLYNELQKD KMAEAYSEIG MKGERRRGKG HDGLYQGLST 660ATKDTYDALH MQALPPR 677The DNA sequence encoding CAR-T20.13 (SEQ ID NO: 30) is as follows:atggccttac cagtgaccgc cttgctcctg ccgctggcct tgctgctcca cgccgccagg 60ccgcaggtgc aattggtgca gtctggcgct gaagttaaga agcctgggag ttcagtgaag 120gtctcctgca aggcttccgg atacgccttc agctattctt ggatcaattg ggtgcggcag 180gcgcctggac aagggctcga gtggatggga cggatctttc ccggcgatgg ggatactgac 240tacaatggga aattcaaggg cagagtcaca attaccgccg acaaatccac tagcacagcc 300tatatggagc tgagcagcct gagatctgag gacacggccg tgtattactg tgcaagaaat 360gtctttgatg gttactggct tgtttactgg ggccagggaa ccctggtcac cgtctcctca 420ggtggcggtg gctcgggcgg tggtgggtcg ggtggcggcg gatctgatat cgtgatgacc 480cagactccac tctccctgcc cgtcacccct ggagagcccg ccagcattag ctgcaggtct 540agcaagagcc tcttgcacag caatggcatc acttatttgt attggtacct gcaaaagcca 600gggcagtctc cacagctcct gatttatcaa atgtccaacc ttgtctctgg cgtccctgac 660cggttctccg gctccgggtc aggcactgat ttcacactga aaatcagcag ggtggaggct 720gaggatgttg gagtttatta ctgcgctcag aatctagaac ttccttacac cttcggcgga 780gggaccaagg tggagatcaa acgtacggtg gagagcaagt acggaccgcc ctgcccccct 840tgccctgccc ccgagttcct gggcggaccc agcgtgttcc tgttcccccc caagcccaag 900gacaccctga tgatcagccg gacccccgag gtgacctgcg tggtggtgga cgtgagccag 960gaagatcccg aggtccagtt caattggtac gtggacggcg tggaagtgca caacgccaag 1020accaagccca gagaggaaca gttcaacagc acctaccggg tggtgtctgt gctgaccgtg 1080ctgcaccagg actggctgaa cggcaaagaa tacaagtgca aggtgtccaa caagggcctg 1140cccagcagca tcgaaaagac catcagcaag gccaagggcc agcctcgcga gccccaggtg 1200tacaccctgc ctccctccca ggaagagatg accaagaacc aggtgtccct gacctgcctg 1260gtgaagggct tctaccccag cgacatcgcc gtggagtggg agagcaacgg ccagcctgag 1320aacaactaca agaccacccc tcccgtgctg gacagcgacg gcagcttctt cctgtacagc 1380cggctgaccg tggacaagag ccggtggcag gaaggcaacg tctttagctg cagcgtgatg 1440cacgaggccc tgcacaacca ctacacccag aagagcctga gcctgtccct gggcaagatc 1500tacatctggg cgcccttggc cgggacttgt ggggtccttc tcctgtcact ggttatcacc 1560ctttactgca aacggggcag aaagaaactc ctgtatatat tcaaacaacc atttatgaga 1620ccagtacaaa ctactcaaga ggaagatggc tgtagctgcc gatttccaga agaagaagaa 1680ggaggatgtg aactgagagt gaagttcagc aggagcgcag acgcccccgc gtacaagcag 1740ggccagaacc agctctataa cgagctcaat ctaggacgaa gagaggagta cgatgttttg 1800gacaagagac gtggccggga ccctgagatg gggggaaagc cgagaaggaa gaaccctcag 1860gaaggcctgt acaatgaact gcagaaagat aagatggcgg aggcctacag tgagattggg 1920atgaaaggcg agcgccggag gggcaagggg cacgatggcc tttaccaggg tctcagtaca 1980gccaccaagg acacctacga cgcccttcac atgcaggccc tgccccctcg ctag 2034CAR-T20.14 (SEQ ID NO: 1):MALPVTALLL PLALLLHAAR PEVQLVESGG GLVQPGRSLR LSCAASGFTF NDYAMHWVRQ 60APGKGLEWVS TISWNSGSIG YADSVKGRFT ISRDNAKKSL YLQMNSLRAE DTALYYCAKD 120IQYGNYYYGM DVWGQGTTVT VSSGGGGSGG GGSGGGGSEI VLTQSPATLS LSPGERATLS 180CRASQSVSSY LAWYQQKPGQ APRLLIYDAS NRATGIPARF SGSGSGTDFT LTISSLEPED 240FAVYYCQQRS NWPITFGQGT RLEIKESKYG PPCPPCPAPE FLGGPSVFLF PPKPKDTLMI 300SRTPEVTCVV VDVSQEDPEV QFNWYVDGVE VHNAKTKPRE EQFNSTYRVV SVLTVLHQDW 360LNGKEYKCKV SNKGLPSSIE KTISKAKGQP REPQVYTLPP SQEEMTKNQV SLTCLVKGFY 420PSDIAVEWES NGQPENNYKT TPPVLDSDGS FFLYSRLTVD KSRWQEGNVF SCSVMHEALH 480NHYTQKSLSL SLGKIYIWAP LAGTCGVLLL SLVITLYCKR GRKKLLYIFK QPFMRPVQTT 540QEEDGCSCRF PEEEEGGCEL RVKFSRSADA PAYKQGQNQL YNELNLGRRE EYDVLDKRRG 600RDPEMGGKPR RKNPQEGLYN ELQKDKMAEA YSEIGMKGER RRGKGHDGLY QGLSTATKDT 660YDALHMQALP PR 672The DNA sequence encoding CAR-T20.14 (SEQ ID NO: 2) is as follows:atggccttac cagtgaccgc cttgctcctg ccgctggcct tgctgctcca cgccgccagg 60ccggaagtgc agctggtgga gtctggggga ggcttggtac agcctggcag gtccctgaga 120ctctcctgtg cagcctctgg attcaccttt aatgattatg ccatgcactg ggtccggcaa 180gctccaggga agggcctgga gtgggtctca actattagtt ggaatagtgg ttccataggc 240tatgcggact ctgtgaaggg ccgattcacc atctccagag acaacgccaa gaagtccctg 300tatctgcaaa tgaacagtct gagagctgag gacacggcct tgtattactg tgcaaaagat 360atacagtacg gcaactacta ctacggtatg gacgtctggg gccaagggac cacggtcacc 420gtctcctcag gtggcggtgg ctcgggcggt ggtgggtcgg gtggcggcgg atctgaaatt 480gtgttgacac agtctccagc caccctgtct ttgtctccag gggaaagagc caccctctcc 540tgcagggcca gtcagagtgt tagcagctac ttagcctggt accaacagaa acctggccag 600gctcccaggc tcctcatcta tgatgcatcc aacagggcca ctggcatccc agccaggttc 660agtggcagtg ggtctgggac agacttcact ctcaccatca gcagcctaga gcctgaagat 720tttgcagttt attactgtca gcagcgtagc aactggccga tcaccttcgg ccaagggaca 780cgactggaga ttaaagagag caagtacgga ccgccctgcc ccccttgccc tgcccccgag 840ttcctgggcg gacccagcgt gttcctgttc ccccccaagc ccaaggacac cctgatgatc 900agccggaccc ccgaggtgac ctgcgtggtg gtggacgtga gccaggaaga tcccgaggtc 960cagttcaatt ggtacgtgga cggcgtggaa gtgcacaacg ccaagaccaa gcccagagag 1020gaacagttca acagcaccta ccgggtggtg tctgtgctga ccgtgctgca ccaggactgg 1080ctgaacggca aagaatacaa gtgcaaggtg tccaacaagg gcctgcccag cagcatcgaa 1140aagaccatca gcaaggccaa gggccagcct cgcgagcccc aggtgtacac cctgcctccc 1200tcccaggaag agatgaccaa gaaccaggtg tccctgacct gcctggtgaa gggcttctac 1260cccagcgaca tcgccgtgga gtgggagagc aacggccagc ctgagaacaa ctacaagacc 1320acccctcccg tgctggacag cgacggcagc ttcttcctgt acagccggct gaccgtggac 1380aagagccggt ggcaggaagg caacgtcttt agctgcagcg tgatgcacga ggccctgcac 1440aaccactaca cccagaagag cctgagcctg tccctgggca agatctacat ctgggcgccc 1500ttggccggga cttgtggggt ccttctcctg tcactggtta tcacccttta ctgcaaacgg 1560ggcagaaaga aactcctgta tatattcaaa caaccattta tgagaccagt acaaactact 1620caagaggaag atggctgtag ctgccgattt ccagaagaag aagaaggagg atgtgaactg 1680agagtgaagt tcagcaggag cgcagacgcc cccgcgtaca agcagggcca gaaccagctc 1740tataacgagc tcaatctagg acgaagagag gagtacgatg ttttggacaa gagacgtggc 1800cgggaccctg agatgggggg aaagccgaga aggaagaacc ctcaggaagg cctgtacaat 1860gaactgcaga aagataagat ggcggaggcc tacagtgaga ttgggatgaa aggcgagcgc 1920cggaggggca aggggcacga tggcctttac cagggtctca gtacagccac caaggacacc 1980tacgacgccc ttcacatgca ggccctgccc cctcgctag 2019CAR-T20.16 (SEQ ID NO: 3)MALPVTALLL PLALLLHAAR PQVQLQQPGA ELVKPGASVK MSCKASGYTF TSYNMHWVKQ 60TPGRGLEWIG AIYPGNGDTS YNQKFKGKAT LTADKSSSTA YMQLSSLTSE DSAVYYCARS 120TYYGGDWYFN VWGAGTTVTV SAGGGGSGGG GSGGGGSQIV LSQSPAILSA SPGEKVTMTC 180RASSSVSYTH WFQQKPGSSP KPWIYATSNL ASGVPVRFSG SGSGTSYSLT ISRVEAEDAA 240TYYCQQWTSN PPTFGGGTKL EIKESKYGPP CPPCPAPEFL GGPSVFLFPP KPKDTLMISR 300TPEVTCVVVD VSQEDPEVQF NWYVDGVEVH NAKTKPREEQ FNSTYRVVSV LTVLHQDWLN 360GKEYKCKVSN KGLPSSIEKT ISKAKGQPRE PQVYTLPPSQ EEMTKNQVSL TCLVKGFYPS 420DIAVEWESNG QPENNYKTTP PVLDSDGSFF LYSRLTVDKS RWQEGNVESC SVMHEALHNH 480YTQKSLSLSL GKIYIWAPLA GTCGVLLLSL VITLYCKRGR KKLLYIFKQP FMRPVQTTQE 540EDGCSCREPE LEIGGCELRV KESRSADAPA YKQGQNQLYN ELNLGRRLEY DVLDKRRGRD 600PEMGGKPRRK NPQEGLYNEL QKDKMAEAYS EIGMKGERRR GKGHDGLYQG LSTATKDTYD 660ALHMQALPPR 670The DNA sequence encoding CAR-T20.16 (SEQ ID NO: 4) is as follows:ATGGCCTTAC CAGTGACCGC CTTGCTCCTG CCGCTGGCCT TGCTGCTCCA CGCCGCCAGG 60CCGCAGGTGC AGTTGCAACA GCCTGGAGCT GAGTTGGTGA AGCCTGGTGC TTCTGTGAAG 120ATGTCTTGTA AGGCTTCTGG ATACACATTC ACTTCTTACA ACATGCACTG GGTGAAGCAG 180ACTCCTGGTA GGGGTTTGGA GTGGATCCGA GCTATCTACC CAGGAAACGG AGACACATCT 240TACAACCAGA AGTTCAAGGG TAAGGCTACA TTGACTGCTG ACAAGTCTTC ATCTACTGCT 300TACATGCAAT TGTCTTCTTT GACATCTGAG GACTCTGCAG TTTACTACTG CGCTAGGTCT 360ACATACTACG GAGGTGACTG GTACTTCAAC GTGTGGGGAG CAGGTACCAC GGTCACTGTC 420TCTGCAGGTG GAGGTGGATC TGGAGGAGGA GGATCTGGTG GAGGAGGTTC TCAAATTGTT 480CTCTCCCAGT CTCCAGCAAT CCTGTCAGCT TCTCCTGGAG AGAAGGTGAC TATGACTTGC 540AGGGCTTCTT CATCTGTTTC TTACATCCAC TGGTTCCAGC AGAAGCCTGG TTCTTCACCT 600AAGCCTTGGA TCTACGCTAC ATCTAACTTG GCATCTGGAG TGCCTGTGAG GTTCTCTGGT 660TCTGGTTCAG GTACTTCTTA CTCTTTGACA ATCTCTAGGG TGGAGGCTGA GGACGCTGCT 720ACTTACTACT GCCAGCAGTG GACATCTAAC CCTCCAACAT TCGGAGGTGG TACTAAGTTG 780GAGATCAAGG AGAGCAAGTA CGGACCGCCC TGCCCCCCTT GCCCTGCCCC CGAGTTCCTG 840GGCGGACCCA GCGTGTTCCT GTTCCCCCCC AAGCCCAAGG ACACCCTGAT GATCAGCCGG 900ACCCCCGAGG TGACCTGCGT GGTGGTGGAC GTGAGCCAGG AAGATCCCGA GGTCCAGTTC 960AATTGGTACG TGGACGGCGT GGAAGTGCAC AACGCCAAGA CCAAGCCCAG AGAGGAACAG 1020TTCAACAGCA CCTACCGGGT GGTGTCTGTG CTGACCGTGC TGCACCAGGA CTGGCTGAAC 1080GGCAAAGAAT ACAAGTGCAA GGTGTCCAAC AAGGGCCTGC CCAGCAGCAT CGAAAAGACC 1140ATCAGCAAGG CCAAGGGCCA GCCTCGCGAG CCCCAGGTGT ACACCCTGCC TCCCTCCCAG 1200GAAGAGATGA CCAAGAACCA GGTGTCCCTG ACCTGCCTGG TGAAGGGCTT CTACCCCAGC 1260GACATCGCCG TGGAGTGGGA GAGCAACGGC CAGCCTGAGA ACAACTACAA GACCACCCCT 1320CCCGTGCTGG ACAGCGACGG CAGCTTCTTC CTGTACAGCC GGCTGACCGT GGACAAGAGC 1380CGGTGGCAGG AAGGCAACGT CTTTAGCTGC AGCGTGATGC ACGAGGCCCT GCACAACCAC 1440TACACCCAGA AGAGCCTGAG CCTGTCCCTG GGCAAGATCT ACATCTGGGC GCCCTTGGCC 1500GGGACTTGTG GGGTCCTTCT CCTGTCACTG GTTATCACCC TTTACTGCAA ACGGGGCAGA 1560AAGAAACTCC TGTATATATT CAAACAACCA TTTATGAGAC CAGTACAAAC TACTCAAGAG 1620GAAGATGGCT GTAGCTGCCG ATTTCCAGAA GAAGAAGAAG GAGGATGTGA ACTGAGAGTG 1680AAGTTCAGCA GGAGCGCAGA CGCCCCCGCG TACAAGCAGG GCCAGAACCA GCTCTATAAC 1740GAGCTCAATC TAGGACGAAG AGAGGAGTAC GATGTTTTGG ACAAGAGACG TGGCCGGGAC 1800CCTGAGATGG GGGGAAAGCC GAGAAGGAAG AACCCTCAGG AAGGCCTGTA CAATGAACTG 1860CAGAAAGATA AGATGGCGGA GGCCTACAGT GAGATTGGGA TGAAAGGCGA GCGCCGGAGG 1920GGCAAGGGGC ACGATGGCCT TTACCAGGGT CTCAGTACAG CCACCAAGGA CACCTACGAC 1980GCCCTTCACA TGCAGGCCCT GCCCCCTCGC TAG 2013

In another more preferred embodiment of the invention, the amino acidsequence of the chimeric antigen receptor (CAR) provided by theinvention is as follows:

CAR-T20.19 (SEQ ID NO: 5)MALPVTALLL PLALLLHAAR PEVQLVESGG GLVQPGRSLR LSCAASGFTF NDYAMHWVRQ 60APGKGLEWVS TISWNSGSIG YADSVKGRFT ISRDNAKKSL YLQMNSLRAE DTALYYCAKD 120IQYGNYYYGM DVWGQGTTVT VSSGGGGSGG GGSGGGGSEI VLTQSPATLS LSPGERATLS 180CRASQSVSSY LAWYQQKPGQ APRLITYDAS NRATGIPARF SGSGSGTDFT LTISSUPED 240FAVYYCQQRS NWPITFGQGT RLEIKESKYG PPCPPCPAPE FEGGPSVFLF PPKPKDTLMI 300SRTPEVTCVV VDVSQEDPEV QFNWYVDGVE VHNAKTKPRE EQFQSTYRVV SVLTVLHQDW 360LNGKEYKCKV SNKGLPSSIE KTISKAKGQP REPQVYTLPP SQEEMTKNQV SLTCLVKGFY 420PSDIAVEWES NGQPENNYKT TPPVLDSDGS FFLYSRLTVD KSRWQEGNVF SCSVMHEALH 480NHYTQKSLSL SLGKIYIWAP LAGTCGVLLL SLVITLYCKR GRKKLLYIFK QPFMRPVQTT 540QEEDGCSCRF PEEEEGGCEL RVKFSRSADA PAYKQGQNQL YNELNLGRRE EYDVLDKRRG 600RDPEMGGKPR RKNPQEGLYN ELQKDKMAEA YSEIGMKGER RRGKGHDGLY QGLSTATKDT 660YDALHMQALP PR 672The DNA sequence encoding CAR-T20.19 (SEQ ID NO: 6) is as follows:atggccttac cagtgaccgc cttgctcctg ccgctggcct tgctgctcca cgccgccagg 60ccggaagtgc agctggtgga gtctggggga ggcttggtac agcctggcag gtccctgaga 120ctctcctgtg cagcctctgg attcaccttt aatgattatg ccatgcactg ggtccggcaa 180gctccaggga agggcctgga gtgggtctca actattagtt ggaatagtgg ttccataggc 240tatgcggact ctgtgaaggg ccgattcacc atctccagag acaacgccaa gaagtccctg 300tatctgcaaa tgaacagtct gagagctgag gacacggcct tgtattactg tgcaaaagat 360atacagtacg gcaactacta ctacggtatg gacgtctggg gccaagggac cacggtcacc 420gtctcctcag gtggcggtgg ctcgggcggt ggtgggtcgg gtggcggcgg atctgaaatt 480gtgttgacac agtctccagc caccctgtct ttgtctccag gggaaagagc caccctctcc 540tgcagggcca gtcagagtgt tagcagctac ttagcctggt accaacagaa acctggccag 600gctcccaggc tcctcatcta tgatgcatcc aacagggcca ctggcatccc agccaggttc 660agtggcagtg ggtctgggac agacttcact ctcaccatca gcagcctaga gcctgaagat 720tttgcagttt attactgtca gcagcgtagc aactggccga tcaccttcgg ccaagggaca 780cgactggaga ttaaagagag caagtacgga ccgccctgcc ccccttgccc tgcccccgag 840ttcgagggcg gacccagcgt gttcctgttc ccccccaagc ccaaggacac cctgatgatc 900agccggaccc ccgaggtgac ctgcgtggtg gtggacgtga gccaggaaga tcccgaggtc 960cagttcaatt ggtacgtgga cggcgtggaa gtgcacaacg ccaagaccaa gcccagagag 1020gaacagttcc aaagcaccta ccgggtggtg tctgtgctga ccgtgctgca ccaggactgg 1080ctgaacggca aagaatacaa gtgcaaggtg tccaacaagg gcctgcccag cagcatcgaa 1140aagaccatca gcaaggccaa gggccagcct cgcgagcccc aggtgtacac cctgcctccc 1200tcccaggaag agatgaccaa gaaccaggtg tccctgacct gcctggtgaa gggcttctac 1260cccagcgaca tcgccgtgga gtgggagagc aacggccagc ctgagaacaa ctacaagacc 1320acccctcccg tgctggacag cgacggcagc ttcttcctgt acagccggct gaccgtggac 1380aagagccggt ggcaggaagg caacgtcttt agctgcagcg tgatgcacga ggccctgcac 1440aaccactaca cccagaagag cctgagcctg tccctgggca agatctacat ctgggcgccc 1500ttggccggga cttgtggggt ccttctcctg tcactggtta tcacccttta ctgcaaacgg 1560ggcagaaaga aactcctgta tatattcaaa caaccattta tgagaccagt acaaactact 1620caagaggaag atggctgtag ctgccgattt ccagaagaag aagaaggagg atgtgaactg 1680agagtgaagt tcagcaggag cgcagacgcc cccgcgtaca agcagggcca gaaccagctc 1740tataacgagc tcaatctagg acgaagagag gagtacgatg ttttggacaa gagacgtggc 1800cgggaccctg agatgggggg aaagccgaga aggaagaacc ctcaggaagg cctgtacaat 1860gaactgcaga aagataagat ggcggaggcc tacagtgaga ttgggatgaa aggcgagcgc 1920cggaggggca aggggcacga tggcctttac cagggtctca gtacagccac caaggacacc 1980tacgacgccc ttcacatgca ggccctgccc cctcgctag 2019

In another most preferred embodiment of the invention, the amino acidsequence of the chimeric antigen receptor (CAR) provided by theinvention is as follows:

CAR-T20.20 (SEQ ID NO: 31)MALPVTALLL PLALLLHAAR PEVQLVESGG GLVQPGRSLR LSCAASGFTF NDYAMHWVRQ 60APGKGLEWVS TISWNSGSIG YADSVKGRFT ISRDNAKKSL YLQMNSLRAE DTALYYCAKD 120IQYGNYYYGM DVWGQGTTVT VSSGGGGSGG GGSGGGGSEI VLTQSPATLS LSPGERATLS 180CRASQSVSSY LAWYQQKPGQ APRLLIYDAS NRATGIPARF SGSGSGTDFT LTISSLEPED 240FAVYYCQQRS NWPITFGQGT RLEIKESKYG PPCPPCPAPE FEGGPSVFLF PPKPKDTLMI 300SRTPEVTCVV VDVSQEDPEV QFNWYVDGVE VHNAKTKPRE EQFQSTYRVV SVLTVLHQDW 360LNGKEYKCKV SNKGLPSSIE KTISKAKGQP REPQVYTLPP SQEEMTKNQV SLTCLVKGFY 420PSDIAVEWES NGQPENNYKT TPPVLDSDGS FFLYSRLTVD KSRWQEGNVF SCSVMHEALH 480NHYTQKSLSL SLGKFWVLVV VGGVLACYSL LVTVAFIIFW VRSKRSRLLH SDYMNMTPRR 540PGPTRKHYQP YAPPRDFAAY RSKRGRKKLL YIFKQPFMRP VQTTQEEDGC SCRFPEEEEG 600GCELRVKFSR SADAPAYKQG QNQLYNELNL GRREEYDVLD KRRGRDPEMG GKPRRKNPQE 660GLYNELQKDK MAEAYSEIGM KGERRRGKGH DGLYQGLSTA TKDTYDALHM QALPPR 716

The coding DNA sequence of CAR-T20.20 (SEQ ID NO: 32) is as follows:

atggccttac cagtgaccgc cttgctcctg ccgctggcct tgctgctcca cgccgccagg 60ccggaagtgc agctggtgga gtctggggga ggcttggtac agcctggcag gtccctgaga 120ctctcctgtg cagcctctgg attcaccttt aatgattatg ccatgcactg ggtccggcaa 180gctccaggga agggcctgga gtgggtctca actattagtt ggaatagtgg ttccataggc 240tatgcggact ctgtgaaggg ccgattcacc atctccagag acaacgccaa gaagtccctg 300tatctgcaaa tgaacagtct gagagctgag gacacggcct tgtattactg tgcaaaagat 360atacagtacg gcaactacta ctacggtatg gacgtctggg gccaagggac cacggtcacc 420gtctcctcag gtggcggtgg ctcgggcggt ggtgggtcgg gtggcggcgg atctgaaatt 480gtgttgacac agtctccagc caccctgtct ttgtctccag gggaaagagc caccctctcc 540tgcagggcca gtcagagtgt tagcagctac ttagcctggt accaacagaa acctggccag 600gctcccaggc tcctcatcta tgatgcatcc aacagggcca ctggcatccc agccaggttc 660agtggcagtg ggtctgggac agacttcact ctcaccatca gcagcctaga gcctgaagat 720tttgcagttt attactgtca gcagcgtagc aactggccga tcaccttcgg ccaagggaca 780cgactggaga ttaaagagag caagtacgga ccgccctgcc ccccttgccc tgcccccgag 840ttcgagggcg gacccagcgt gttcctgttc ccccccaagc ccaaggacac cctgatgatc 900agccggaccc ccgaggtgac ctgcgtggtg gtggacgtga gccaggaaga tcccgaggtc 960cagttcaatt ggtacgtgga cggcgtggaa gtgcacaacg ccaagaccaa gcccagagag 1020gaacagttcc aaagcaccta ccgggtggtg tctgtgctga ccgtgctgca ccaggactgg 1080ctgaacggca aagaatacaa gtgcaaggtg tccaacaagg gcctgcccag cagcatcgaa 1140aagaccatca gcaaggccaa gggccagcct cgcgagcccc aggtgtacac cctgcctccc 1200tcccaggaag agatgaccaa gaaccaggtg tccctgacct gcctggtgaa gggcttctac 1260cccagcgaca tcgccgtgga gtgggagagc aacggccagc ctgagaacaa ctacaagacc 1320acccctcccg tgctggacag cgacggcagc ttcttcctgt acagccggct gaccgtggac 1380aagagccggt ggcaggaagg caacgtcttt agctgcagcg tgatgcacga ggccctgcac 1440aaccactaca cccagaagag cctgagcctg tccctgggca agttttgggt gctggtggtg 1500gttggtggag tcctggcttg ctatagcttg ctagtaacag tggcctttat tattttctgg 1560gtgaggagta agaggagcag gctcctgcac agtgactaca tgaacatgac tccccgccgc 1620cccgggccca cccgcaagca ttaccagccc tatgccccac cacgcgactt cgcagcctat 1680cgctccaaac ggggcagaaa gaaactcctg tatatattca aacaaccatt tatgagacca 1740gtacaaacta ctcaagagga agatggctgt agctgccgat ttccagaaga agaagaagga 1800ggatgtgaac tgagagtgaa gttcagcagg agcgcagacg cccccgcgta caagcagggc 1860cagaaccagc tctataacga gctcaatcta ggacgaagag aggagtacga tgttttggac 1920aagagacgtg gccgggaccc tgagatgggg ggaaagccga gaaggaagaa ccctcaggaa 1980ggcctgtaca atgaactgca gaaagataag atggcggagg cctacagtga gattgggatg 2040aaaggcgagc gccggagggg caaggggcac gatggccttt accagggtct cagtacagcc 2100accaaggaca cctacgacgc ccttcacatg caggccctgc cccctcgcta a 2151

Antigen Binding Domain

In one embodiment, the CAR of the invention comprises a target-specificbinding element referred to as antigen binding domain. The antigenbinding domain of the CAR of the invention is a specific binding elementtargeting CD20.

In a preferred embodiment of the invention, the antigen binding domaincomprises a heavy chain variable region and a light chain variableregion of an anti-CD20 antibody.

In another preferred embodiment, the amino acid sequence of the heavychain variable region of Ofatumumaband antibody is as follows:

-   -   EVQLVESGGG LVQPGRSLRL SCAASGFTFN DYAMHWVRQA PGKGLEWVST        ISWNSGSIGY 60 ADSVKGRFTI SRDNAKKSLY LQMNSLRAED TALYYCAKDI        QYGNYYYGMD VWGQGTTVTV 120 SS 122 (SEQ ID NO:7);

and the DNA sequence encoding the heavy chain variable region ofOfatumumaband antibody is as follows:

(SEQ ID NO: 8)GAAGTGCAGC TGGTGGAGTC TGGGGGAGGC TTGGTACAGC CTGGCAGGTC CCTGAGACTC  60TCCTGTGCAG CCTCTGGATT CACCTTTAAT GATTATGCCA TGCACTGGGT CCGGCAAGCT 120CCAGGGAAGG GCCTGGAGTG GGTCTCAACT ATTAGTTGGA ATAGTGGTTC CATAGGCTAT 180GCGGACTCTG TGAAGGGCCG ATTCACCATC TCCAGAGACA ACGCCAAGAA GTCCCTGTAT 240CTGCAAATGA ACAGTCTGAG AGCTGAGGAC ACGGCCTTGT ATTACTGTGC AAAAGATATA 300CAGTACGGCA ACTACTACTA CGGTATGGAC GTCTGGGGCC AAGGGACCAC GGTCACCGTC 360TCCTCA;                                                           366

or,

the amino acid sequence of the heavy chain variable region of Rituximabantibody is as follows:

(SEQ ID NO: 9)QVQLQQPGAE LVKPGASVKM SCKASGYTFT SYNMHWVKQT PGRGLEWIGA IYPGNGDTSY  60NQKFKGKATL TADKSSSTAY MQLSSLTSED SAVYYCARST YYGGDWYFNV WGAGTTVTVS 120A;                                                                121

and the DNA sequence encoding the heavy chain variable region ofRituximab antibody is as follows:

(SEQ ID NO: 10)CAGGTGCAGT TGCAACAGCC TGGAGCTGAG TTGGTGAAGC CTGGTGCTTC TGTGAAGATG  60TCTTGTAAGG CTTCTGGATA CACATTCACT TCTTACAACA TGCACTGGGT GAAGCAGACT 120CCTGGTAGGG GTTTGGAGTG GATCGGAGCT ATCTACCCAG GAAACGGAGA CACATCTTAC 180AACCAGAAGT TCAAGGGTAA GGCTACATTG ACTGCTGACA AGTCTTCATC TACTGCTTAC 240ATGCAATTGT CTTCTTTGAC ATCTGAGGAC TCTGCAGTTT ACTACTGCGC TAGGTCTACA 300TACTACGGAG GTGACTGGTA CTTCAACGTG TGGGGAGCAG GTACCACGGT CACTGTCTCT 360GCA.                                                              363

Further, the amino acid sequence of the heavy chain variable region ofObinutuzumab antibody used in the present invention is as follows:

(SEQ ID NO: 33)QVQLVQSGAE VKKPGSSVKV SCKASGYAFS YSWINWVRQA PGQGLEWMGR IFPGDGDTDY 60NGKFKGRVTI TADKSTSTAY MELSSLRSED TAVYYCARNV FDGYWLVYWG QGTLVTVSS 119

The DNA sequence encoding the heavy chain variable region ofObinutuzumab antibody is as follows:

(SEQ ID NO: 34)caggtgcaat tggtgcagtc tggcgctgaa gttaagaagc ctgggagttc agtgaaggtc  60tcctgcaagg cttccggata cgccttcagc tattcttgga tcaattgggt gcggcaggcg 120cctggacaag ggctcgagtg gatgggacgg atctttcccg gcgatgggga tactgactac 180aatgggaaat tcaagggcag agtcacaatt accgccgaca aatccactag cacagcctat 240atggagctga gcagcctgag atctgaggac acggccgtgt attactgtgc aagaaatgtc 300tttgatggtt actggcttgt ttactggggc cagggaaccc tggtcaccgt ctcctca    357

In another preferred embodiment, the amino acid sequence of the lightchain variable region of Ofatumumaband antibody is as follows:

(SEQ ID NO: 11)EIVLTQSPAT LSLSPGERAT LSCRASQSVS SYLAWYQQKP GQAPRLLTYD ASNRATGIPA  60RFSCSGSGTD FTLTISSLEP EDFAVYYCQQ RSNWPITFGQ GTRLEIK               107

The DNA sequence of Ofatumumaband antibody is as follows:

(SEQ ID NO: 12)GAAATTGTGT TGACACAGTC TCCAGCCACC CTGTCTTTGT CTCCAGGGGA AAGAGCCACC  60CTCTCCTGCA GGGCCAGTCA GAGTGTTAGC AGCTACTTAG CCTGGTACCA ACAGAAACCT 120GGCCAGGCTC CCAGGCTCCT CATCTATGAT GCATCCAACA GGGCCACTGG CATCCCAGCC 180AGGTTCAGTG GCAGTGGGTC TGGGACAGAC TTCACTCTCA CCATCAGCAG CCTAGAGCCT 240GAAGATTTTG CAGTTTATTA CTGTCAGCAG CGTAGCAACT GGCCGATCAC CTTCGGCCAA 300GGGACACGAC TGGAGATTAA A                                           321

Alternatively, the amino acid sequence of the light chain variableregion of Rituximab antibody is as follows:

(SEQ ID NO: 13)QIVLSQSPAI LSASPGEKVT MTCRASSSVS YTHWFQQKPG SSPKPWIYAT SNLASGVPVR  60FSGSGSGTSY SLTISRVEAE DAATYYCQQW TSNPPTFGGG TKLEIK                106

The DNA sequences encoding the light chain (V_(L)) of single-chainvariable region derived from Rituximab antibody is:

(SEQ ID NO:14)CAAATTGTTC TCTCCCAGTC TCCAGCAATC CTGTCAGCTT CTCCTGGAGA GAAGGTGACT  60ATGACTTGCA GGGCTTCTTC ATCTGTTTCT TACATCCACT GGTTCCAGCA GAAGCCTGGT 120TCTTCACCTA AGCCTTGGAT CTACGCTACA TCTAACTTGG CATCTGGAGT GCCTGTGAGG 180TTCTCTGGTT CTGGTTCAGG TACTTCTTAC TCTTTGACAA TCTCTAGGGT GGAGGCTGAG 240GACGCTGCTA CTTACTACTG CCAGCAGTGG ACATCTAACC CTCCAACATT CGGAGGTGGT 300ACTAAGTTGC AGATCAAC.                                              318

Further, the amino acid sequence of the light chain variable region ofObinutuzumab antibody used in the present invention is as follows:

(SEQ ID NO:35)DIVMTQTPLS LPVTPGEPAS ISCRSSKSLL HSNGITYLYW YLQKPGQSPQ LLIYQMSNLV  60SGVPDRFSGS GSGTDFTLKI SRVEAEDVGV YYCAQNLELP YTFGGGTKVE IKRTV      115

The DNA sequence encoding the heavy chain variable region ofObinutuzumab antibody is as follows:

(SEQ ID NO: 36)gatatcgtga tgacccagac tccactctcc ctgcccgtca cccctggaga gcccgccagc  60attagctgca ggtctagcaa gagcctcttg cacagcaatg gcatcactta tttgtattgg 120tacctgcaaa agccagggca gtctccacag ctcctgattt atcaaatgtc caaccttgtc 180tctggcgtcc ctgaccggtt ctccggctcc gggtcaggca ctgatttcac actgaaaatc 240agcagggtgg aggctgagga tgttggagtt tattactgcg ctcagaatct agaacttcct 300tacaccttcg gcggagggac caaggtggag atcaaacgta cggtg                 345

In a preferred embodiment of the invention, the amino acid sequence ofthe linker between the heavy chain variable region and the light chainvariable region is as follows:

(SEQ ID NO: 15) GGGGSGGGGS GGGGS 15

and its coding DNA sequence is as follows:

(SEQ ID NO: 16) GGTGGCGGTG GCTCGGGCGG TGGTGGGTCG GGTGGCGGCG GATCT 45

Hinge Region and Transmembrane Region

As for the hinge region and the transmembrane region (transmembranedomain), the CAR can be designed to comprise a transmembrane domainfused to the extracellular domain of the CAR. In one embodiment, atransmembrane domain that is naturally associated with one of thedomains in the CAR is used. In some embodiments, transmembrane domainsmay be selected or modified by amino acid substitutions to avoid bindingsuch domains to the transmembrane domain of the same or differentsurface membrane proteins, thereby minimizing the interaction with othermembers of the receptor complexes.

In a preferred embodiment of the invention, the hinge region comprisesthe following amino acid sequence (IgG4 Hinge-CH2-CH3 hinge region):

(SEQ ID NO: 17)ESKYGPPCPP CPAPEFLGGP SVFLFPPKPK DTLMISRTPE VTCVVVDVSQ EDPEVQFNWY  60VDGVEVHNAK TKPREEQFNS TYRVVSVLTV LHQDWLNGKE YKCKVSNKGL PSSIEKTISK 120AKGQPREPQV YTLPPSQEEM TKNQVSLTCL VKGFYPSDIA VEWESNGQPE NNYKTTPPVL 180DSDGSFFLYS RLTVDKSRWQ EGNVFSCSVM HEALHNHYTQ KSLSLSLGK             229

Its coding DNA sequence is as follows:

(SEQ ID NO: 18)GAGAGCAAGT ACGGACCGCC CTGCCCCCCT TGCCCTGCCC CCGAGTTCCT GGGCGGACCC  60AGCGTGTTCC TGTTCCCCCC CAAGCCCAAG GACACCCTGA TGATCAGCCG GACCCCCGAG 120GTGACCTGCG TGGTGGTGGA CGTGAGCCAG GAAGATCCCG AGGTCCAGTT CAATTGGTAC 180GTGGACGGCG TGGAAGTGCA CAACGCCAAG ACCAAGCCCA GAGAGGAACA GTTCAACAGC 240ACCTACCGGG TGGTCTCTGT GCTGACCGTG CTGCACCAGG ACTGGCTGAA CGGCAAAGAA 300TACAAGTGCA AGGTGTCCAA CAAGGGCCTG CCCAGCAGCA TCGAAAAGAC CATCAGCAAG 360GCCAAGGGCC AGCCTCGCGA GCCCCAGGTG TACACCCTGC CTCCCTCCCA GGAAGAGATG 420ACCAAGAACC AGGTGTCCCT GACCTGCCTG GTGAAGGGCT TCTACCCCAG CGACATCGCC 480GTGGAGTGGG AGAGCAACGG CCAGCCTGAG AACAACTACA AGACCACCCC TCCCGTGCTG 540GACAGCGACG GCAGCTTCTT CCTGTACAGC CGGCTGACCG TGGACAAGAG CCGGTGGCAG 600GAAGGCAACG TCTTTAGCTG CAGCGTGATG CACGAGGCCC TGCACAACCA CTACACCCAG 660AAGAGCCTGA GCCTGTCCCT GGGCAAG 687;

or, the hinge region comprises the following amino acid sequence (IgG4Hinge-C112-CH3 (L235E, N297Q)):

(SEQ ID NO: 19)ESKYGPPCPP CPAPEFEGGP SVFLFPPKPK DTLMISRTPE VTCVVVDVSQ EDPEVQFNWY  60VDGVEVHNAK TKPREEQFQS TYRVVSVLTV LHQDWLNGKE YKCKVSNKGL PSSTEKTTSK 120AKGQPREPQV YTLPPSQEEM TKNQVSLTCL VKGFYPSDIA VEWESNGQPE NNYKTTPPVL 180DSDGSFFLYS RLTVDKSRWQ EGNVFSCSVM HEALHNHYTQ KSLSLSLGK 229

Its coding DNA sequence is as follows:

(SEQ ID NO: 20)GAGAGCAAGT ACGGACCGCC CTGCCCCCCT TGCCCTGCCC CCGAGTTCGA GGGCGGACCC  60AGCGTGTTCC TGTTCCCCCC CAAGCCCAAG GACACCCTGA TGATCAGCCG GACCCCCGAG 120GTGACCTGCG TGGTGGTGGA CGTGAGCCAG GAAGATCCCG AGGTCCAGTT CAATTGGTAC 180GTGGACGGCG TGGAAGTGCA CAACGCCAAG ACCAAGCCCA GAGAGGAACA GTTCCAAAGC 240ACCTACCGGG TGGTGTCTGT GCTGACCGTG CTGCACCAGG ACTGGCTGAA CGGCAAAGAA 300TACAAGTGCA AGGTGTCCAA CAAGGGCCTG CCCAGCAGCA TCGAAAAGAC CATCAGCAAG 360GCCAAGGGCC AGCCTCGCGA GCCCCAGGTG TACACCCTGC CTCCCTCCCA GGAAGAGATG 420ACCAAGAACC AGGTGTCCCT GACCTGCCTG GTGAAGGGCT TCTACCCCAG CGACATCGCC 480GTGGAGTGGG AGAGCAACGG CCAGCCTGAG AACAACTACA AGACCACCCC TCCCGTGCTG 540GACAGCGACG GCAGCTTCTT CCTGTACAGC CGGCTGACCG TGGACAAGAG CCGGTGGCAG 600GAAGGCAACG TCTTTAGCTG CAGCGTGATG CACGAGGCCC TGCACAACCA CTACACCCAG 660AAGAGCCTGA GCCTGTCCCT GGGCAAG 687.

In a preferred embodiment of the invention, the amino acid sequence ofthe transmembrane region derived from CD8 (CD8TM) is as follows:

(SEQ ID NO: 21) IYIWAPLAGT CGVLLLSLVI TLYC 24

The coding DNA sequence thereof is as follows:

(SEQ ID NO: 22)ATCTACATCT GGGCGCCCTT GGCCGGGACT TGTGGGGTCC TTCTCCTGTC ACTGGTTATC 60ACCCTTTACT GC 72

In a preferred embodiment of the invention, the amino acid sequence ofthe transmembrane region derived from CD28 (CD28TM) is as follows:

(SEQ ID NO: 37) FWVLVVVGGV LACYSLLVTV AFIIFWV 27;

The DNA sequence encoding the transmembrane region derived from CD28(CD28TM) is as follows:

(SEQ ID NO: 38)TTTTGGGTGC TGGTGGTGGT TGGTGGAGTC CTGGCTTGCT ATAGCTTGCT AGTAACAGTG 60GCCTTTATTA TTTTCTGGGT G 81.

Intracellular Domain

The intracellular domain in the CAR of the invention comprises thesignaling domain of 4-1BB and the signaling domain of CD3ζ.

Preferably, the intracellular signaling domain of 4-1BB comprises thefollowing amino acid sequence:

(SEQ ID NO: 23) KRGRKKLLYI FKQPFMRPVQ TTQEEDGCSC RFPEEEEGGC EL 42

The coding DNA sequence thereof is as follows:

(SEQ ID NO: 24)AAACGGGGCA GAAAGAAACT CCTGTATATA TTCAAACAAC CATTTATGAG ACCAGTACAA  60ACTACTCAAG AGGAAGATGG CTGTAGCTGC CGATTTCCAG AAGAAGAAGA AGGAGGATGT 120GAACTG 126

Preferably, the intracellular signaling domain derived from CD28comprises the following amino acid sequence:

(SEQ ID NO: 39) RSKRSRLLHS DYMNMTPRRP GPTRKHYQPY APPRDFAAYR S 41

The coding DNA sequence thereof is as follows:

(SEQ ID NO: 40)AGGAGTAAGA GGAGCAGGCT CCTGCACAGT GACTACATGA ACATGACTCC CCGCCGCCCC  60GGGCCCACCC GCAAGCATTA CCAGCCCTAT GCCCCACCAC GCGACTTCGC AGCCTATCGC 120TCC 123

Preferably, the intracellular signaling domain of CD3ζ comprises thefollowing amino acid sequence:

(SEQ ID NO: 25)RVKFSRSADA PAYQQGQNQL YNELNLGRRE EYDVLDKRRG RDPEMGGKPQ RRKNPQEGLY  60NELQKDKMAE AYSEIGMKGE RRRGKGHDGL YQGLSTATKD TYDALHMQAL PPR 113

The coding DNA sequence thereof is as follows:

(SEQ ID NO: 26)AGAGTGAAGT TCAGCAGGAG CGCAGACGCC CCCGCGTACA AGCAGGGGCA GAACCAGCTC  60TATAACGAGC TCAATCTAGG ACGAAGAGAG GAGTACGATG TTTTGGACAA GAGACGTGGC 120CGGGACCCTG AGATGGGGGG AAAGCCGAGA AGGAAGAACC CTCAGGAAGG CCTGTACAAT 180GAACTGCAGA AAGATAAGAT GGCGGAGGCC TACAGTGAGA TTGGGATGAA AGGCGAGCGC 240CGGAGGGGCA AGGGGCACGA TGGCCTTTAC CAGGGTCTCA GTACAGCCAC CAAGGACACC 300TACGACGCCC TTCACATGCA GGCCCTGCCC CCTCGC 336

Vector

The invention also provides a DNA construct encoding the CAR sequencesof the invention.

The nucleic acid sequences coding for the desired molecules can beobtained using recombinant methods known in the art, such as, forexample by screening libraries from cells expressing the gene, byderiving the gene from a vector known to include the same, or byisolating directly from cells and tissues containing the same, usingstandard techniques. Alternatively, the gene of interest can be producedsynthetically.

The present invention also provides vectors in which the DNA constructof the present invention is inserted. Vectors derived from retrovirusessuch as the lentivirus are suitable tools to achieve long-term genetransfer since they allow long-term, stable integration of a transgeneand its propagation in daughter cells. Lentiviral vectors have theadvantage over vectors derived from onco-retroviruses such as murineleukemia viruses in that they can transduce non-proliferating cells,such as hepatocytes. They also have the advantage of low immunogenicity.

In brief summary, the expression of natural or synthetic nucleic acidsencoding CARs is typically achieved by operably linking a nucleic acidencoding the CAR polypeptide or portions thereof to a promoter, andincorporating the construct into an expression vector. The vectors canbe suitable for replication and integration in eukaryotes. Typicalcloning vectors contain transcription and translation terminators,initiation sequences, and promoters useful for regulation of theexpression of the desired nucleic acid sequence.

The expression constructs of the present invention may also be used fornucleic acid immune and gene therapy, using standard gene deliveryprotocols. Methods for gene delivery are known in the art. See, e.g.,U.S. Pat. Nos. 5,399,346, 5,580,859, 5,589,466, incorporated byreference herein in their entireties. In another embodiment, theinvention provides a gene therapy vector,

The nucleic acid can be cloned into a number of types of vectors. Forexample, the nucleic acid can be cloned into a vector including, but notlimited to a plasmid, a phagemid, a phage derivative, an animal virus,and a cosmid. Vectors of particular interest include expression vectors,replication vectors, probe generation vectors, and sequencing vectors,

Further, the expression vector may be provided to a cell in the form ofa viral vector. Viral vector technology is well known in the art and isdescribed, for example, in Sambrook et al, (2001, Molecular Cloning: ALaboratory Manual, Cold Spring Harbor Laboratory, New York), and inother virology and molecular biology manuals. Viruses, which are usefulas vectors include, but are not limited to, retroviruses, adenoviruses,adeno-associated viruses, herpes viruses, and lentiviruses. In general,a suitable vector contains an origin of replication functional in atleast one organism, a promoter sequence, convenient restrictionendonuclease sites, and one or more selectable markers, (e.g., WO01/96584; WO 01/29058; and U.S. Pat. No. 6,326,193).

A number of viral based systems have been developed for gene transferinto mammalian cells. For example, retroviruses provide a convenientplatform for gene delivery systems. A selected gene can be inserted intoa vector and packaged in retroviral particles using techniques known inthe art. The recombinant virus can then be isolated and delivered tocells of the subject either in vivo or ex vivo. A number of retroviralsystems are known in the art. In some embodiments, adenovirus vectorsare used. A number of adenovirus vectors are known in the art. In oneembodiment, lentivirus vectors are used.

Additional promoter elements, e.g., enhancers, regulate the frequency oftranscriptional initiation. Typically, these are located in the region30-110 bp upstream of the start site, although a number of promotershave recently been shown to contain functional elements downstream ofthe start site as well. The spacing between promoter elements frequentlyis flexible, so that promoter function is preserved when elements areinverted or moved relative to one another. In the thymidine kinase (tk)promoter, the spacing between promoter elements can be increased to 50bp apart before activity begins to decline. Depending on the promoter,it appears that individual elements can function either cooperatively orindependently to activate transcription.

One example of a suitable promoter is the immediate earlycytomegalovirus (CMV) promoter sequence. This promoter sequence is astrong constitutive promoter sequence capable of driving high levels ofexpression of any polynucleotide sequence operatively linked thereto.Another example of a suitable promoter is Elongation Growth Factor-1α(EF-1α). However, other constitutive promoter sequences may also beused, including, but not limited to the simian virus 40 (SV40) earlypromoter, mouse mammary tumor virus (MMTV), human immunodeficiency virus(HIV) long terminal repeat (LTR) promoter, MoMuLV promoter, an avianleukemia virus promoter, an Epstein-Barr virus immediate early promoter,a Rous sarcoma virus promoter, as well as human gene promoters such as,but not limited to, the actin promoter, the myosin promoter, thehemoglobin promoter, and the creatine kinase promoter. Further, theinvention should not be limited to the use of constitutive promoters,inducible promoters are also contemplated as part of the invention. Theuse of an inducible promoter provides a molecular switch capable ofturning on expression of the polynucleotide sequence which it isoperatively linked when such expression is desired, or turning off theexpression when expression is not desired. Examples of induciblepromoters include, but are not limited to a metallothionein promoter, aglucocorticoid promoter, a progesterone promoter, and a tetracyclinepromoter.

In order to assess the expression of a CAR polypeptide or portionsthereof, the expression vector to be introduced into a ceil can alsocontain either a selectable marker gene or a reporter gene or both tofacilitate identification and selection of expressing cells from thepopulation of cells sought to be transfected or infected through viralvectors. In other aspects, the selectable marker may be carried on aseparate piece of DNA and used in a co-transfection procedure. Bothselectable markers and reporter genes may be flanked with appropriateregulatory sequences to enable expression in the host cells. Usefulselectable markers include, for example, antibiotic-resistance genes,such as neo and the like.

Reporter genes are used for identifying potentially transfected cellsand for evaluating the functionality of regulatory sequences. Ingeneral, a reporter gene is a gene that is not present in or expressedby the recipient organism or tissue and that encodes a polypeptide whoseexpression is manifested by some easily detectable property, e.g.,enzymatic activity. Expression of the reporter gene is assayed at asuitable time after the DNA has been introduced into the recipientcells. Suitable reporter genes may include genes encoding luciferase,beta-galactosidase, chloramphenicol acetyl transferase, secretedalkaline phosphatase, or the green fluorescent protein gene (e.g.,Ui-Tei et al., 2000 FEBS Letters 479: 79-82). Suitable expressionsystems are well known and may be prepared using known techniques orobtained commercially. In general, the construct with the minimal 5′flanking region showing the highest level of expression of reporter geneis identified as the promoter. Such promoter regions may be linked to areporter gene and used to evaluate agents for the ability to modulatepromoter-driven transcription.

Methods of introducing and expressing genes into a cell are known in theart. In the context of an expression vector, the vector can be readilyintroduced into a host cell, e.g., mammalian, bacterial, yeast, orinsect cell by any method in the art. For example, the expression vectorcan be transferred into a host cell by physical, chemical, or biologicalmeans.

Physical methods for introducing a polynucleotide into a host cellinclude calcium phosphate precipitation, lipofection, particlebombardment, microinjection, electroporation, and the like. Methods forproducing cells comprising vectors and/or exogenous nucleic acids arewell-known in the art. See, for example, Sambrook et al. (2001,Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory,New York). A preferred method for the introduction of a polynucleotideinto a host cell is calcium phosphate transfection.

Biological methods for introducing a polynucleotide of interest into ahost cell include the use of DNA and RNA vectors. Viral vectors, andespecially retroviral vectors, have become the most widely used methodfor inserting genes into mammalian, e.g., human cells. Other viralvectors can be derived from lentivirus, poxviruses, herpes simplex virusI, adenoviruses and adeno-associated viruses, and the like. See, forexample, U.S. Pat. Nos. 5,350,674 and 5,585,362.

Chemical means for introducing a polynucleotide into a host cell includecolloidal dispersion systems, such as macromolecule complexes,nanocapsules, microspheres, beads, and lipid-based systems includingoil-in-water emulsions, micelles, mixed micelles, and liposomes. Anexemplary colloidal system for use as a delivery vehicle in vitro and invivo is a liposome (e.g., an artificial membrane vesicle).

In the case where a non-viral delivery system is utilized, an exemplarydelivery vehicle is a liposome. The use of lipid formulations iscontemplated for the introduction of the nucleic acids into a host cell(in vitro, ex vivo or in vivo). In another aspect, the nucleic acid maybe associated with a lipid. The nucleic acid associated with a lipid maybe encapsulated in the aqueous interior of a liposome, interspersedwithin the lipid bilayer of a liposome, attached to a liposome via alinking molecule that is associated with both the liposome and theoligonucleotide, entrapped in a liposome, complexed with a liposome,dispersed in a solution containing a lipid, mixed with a lipid, combinedwith a lipid, contained as a suspension in a lipid, contained orcomplexed with a micelle, or otherwise associated with a lipid. Lipid,lipid/DNA or lipid/expression vector associated compositions are notlimited to any particular structure in solution. For example, they maybe present in a bilayer structure, as micelles, or with a “collapsed”structure. They may also simply be interspersed in a solution, possiblyforming aggregates that are not uniform in size or shape. Lipids arefatty substances which may be naturally occurring or synthetic lipids.For example, lipids include the fatty droplets that naturally occur inthe cytoplasm as well as the class of compounds which contain long-chainaliphatic hydrocarbons and their derivatives, such as fatty acids,alcohols, amines, amino alcohols, and aldehydes.

In the case where a non-viral delivery system is utilized, genomeediting technique is exemplarily employed to complete the invention, forexample CRISPR-Cas9, ZFN or TALEN.

In a preferred embodiment of the invention, the vector is a lentiviralvector.

In a preferred embodiment of the invention, the DNA construct furthercomprises a signal peptide coding sequence. Preferably, the signalpeptide sequence is ligated upstream of the nucleic acid sequence ofantigen binding domain. Preferably the signal peptide is a human CD8asignal peptide.

Preferably, the amino acid sequence of the signal peptide is as follows:

The amino acid sequence of CD8 leader sequence is:

(SEQ ID NO: 27) MALPVTALL PLALLLHAAR P 21

The DNA sequence encoding CD8 leader sequence is:

(SEQ ID NO: 28)ATGGCCTTAC CAGTGACCGC CTTGCTCCTG CCGCTGGCCT TGCTGCTCCA CGCCGCCAGG 60 CCG63

Therapeutic Application

The present invention encompasses a cell (e.g., T cell) transduced witha lentiviral vector (LV) encoding the CAR of the invention. Thetransduced T cell can elicit a CAR-mediated T-cell response.

Thus, the present invention also provides a method for stimulating a Tcell-mediated immune response to a target cell population or tissue in amammal comprising the step of administering to the mammal a T cell thatexpresses the CAR of the invention.

In one embodiment, the present invention includes a type of cellulartherapy where T cells are genetically modified to express the CAR of theinvention and the CAR-T cell is infused to a recipient in need thereof.The infused cell is able to kill tumor cells in the recipient. Unlikeantibody therapies, CAR-T cells are able to replicate in vivo resultingin long-term persistence that can lead to sustained tumor control.

In one embodiment, the CAR-T cells of the invention can undergo robustin vivo T cell expansion and can persist for an extended amount of time.In addition, the CAR mediated immune response may be part of an adoptiveimmunotherapy approach in which CAR-modified T cells induce an immuneresponse specific to the antigen binding moiety in the CAR. For example,an anti-CD20 CAR-T cell elicits an immune response specific againstcells expressing CD20.

Although the data disclosed herein specifically disclose lentiviralvector comprising anti-CD20 scFv, hinge and transmembrane domain, and4-1BB and CD3ζ signaling domains, the invention should be construed toinclude any number of variations for each of the components of theconstruct as described elsewhere herein.

Adaptation diseases that may be treated include CD20 positive tumors anddiseases caused by excessive B cells (such as autoimmune diseases, forexample, lupus erythematosus, etc.). CD20 positive tumors may includeCD20 positive non-solid tumors (such as hematological tumors, forexample, leukemias and lymphomas) or solid tumors. Types of tumors orcancers to be treated with the CARs of the invention include, but arenot limited to, carcinoma, blastoma, and sarcoma, and certain leukemiaor lymphoid malignancies, benign and malignant tumors, and malignanciese.g., sarcomas, carcinomas, and melanomas. Adult tumors/cancers andpediatric tumors/cancers are also included.

Hematologic cancers are cancers of the blood or bone marrow. Examples ofhematological (or hematogenous) cancers include leukemias, includingacute leukemias (such as acute lymphocytic leukemia, acute myelocyticleukemia, acute myelogenous leukemia and myeloblasts, promyeiocytic,myelomonocytic, monocytic and erythroleukemia), chronic leukemias (suchas chronic myelocytic (granulocytic) leukemia, chronic myelogenousleukemia, and chronic lymphocytic leukemia), polycythemia vera,lymphoma, Hodgkin's disease, non-Hodgkin's lymphoma (indolent and highgrade forms), multiple myeloma, Waldenstrom's macroglobulinemia, heavychain disease, myelodysplastic syndrome, hairy cell leukemia andmyelodysplasia.

Solid tumors are abnormal masses of tissue that usually do not containcysts or liquid areas. Solid tumors can be benign or malignant.Different types of solid tumors are named for the type of cells thatform them (such as sarcomas, carcinomas, and lymphomas). Examples ofsolid tumors, such as sarcomas and carcinomas, include fibrosarcoma,myxosarcoma, liposarcoma, mesothelioma, malignant lymphoma, pancreaticcancer and ovarian cancer.

The CAR-modified T cells of the invention may also serve as a type ofvaccine for ex vivo immunization and/or in vivo therapy in a mammal.Preferably, the mammal is a human.

With respect to ex vivo immunization, at least one of the followingoccurs in vitro prior to administering the cell into a mammal: i)expansion of the cells, ii) introducing a nucleic acid encoding a CAR tothe cells, and/or iii) cryopreservation of the cells.

Ex vivo procedures are well known in the art and are discussed morefully below. Briefly, cells are isolated from a mammal (preferably ahuman) and genetically modified (i.e., transduced or transfected invitro) with a vector expressing a CAR disclosed herein. The CAR-modifiedcell can be administered to a mammalian recipient to provide atherapeutic benefit. The mammalian recipient may be a human and theCAR-modified cell can be autologous with respect to the recipient.Alternatively, the cells can be allogeneic, syngeneic or xenogeneic withrespect to the recipient.

In addition to using a cell-based vaccine in terms of ex vivoimmunization, the present invention also provides compositions andmethods for in vivo immunization to elicit an immune response directedagainst an antigen in a patient.

Generally, the cells activated and expanded as described herein may beutilized in the treatment and prevention of diseases that arise inindividuals who are immunocompromised. In particular, the CAR-modified Tcells of the invention are used in the treatment of CCL. In certainembodiments, the cells of the invention are used in the treatment ofpatients at risk for developing CCL. Thus, the present inventionprovides methods for the treatment or prevention of CCL comprisingadministering to a subject in need thereof, a therapeutically effectiveamount of the CAR-modified T cells of the invention.

The CAR-modified T cells of the present invention may be administeredeither alone, or as a pharmaceutical composition in combination withdiluents and/or with other components such as IL-2, IL-17 or othercytokines or cell populations. Briefly, pharmaceutical compositions ofthe present invention may comprise a target cell population as describedherein, in combination with one or more pharmaceutically orphysiologically acceptable carriers, diluents or excipients. Suchcompositions may comprise buffers such as neutral buffered saline,phosphate buffered saline and the like; carbohydrates such as glucose,mannose, sucrose or dextrans, mannitol; proteins; polypeptides or aminoacids such as glycine; antioxidants; chelating agents such as EDTA orglutathione; adjuvants (e.g., aluminum hydroxide); and preservatives.Compositions of the present invention are preferably formulated forintravenous administration.

Pharmaceutical compositions of the present invention may be administeredin a manner appropriate to the disease to be treated (or prevented). Thequantity and frequency of administration will be determined by suchfactors as the condition of the patient, and the type and severity ofthe patient's disease, although appropriate dosages may be determined byclinical trials.

When “an immunologically effective amount”, “an anti-tumor effectiveamount”, “an tumor-inhibiting effective amount”, or “therapeutic amount”is indicated, the precise amount of the compositions of the presentinvention to be administered can be determined by a physician withconsideration of individual differences in age, weight, tumor size,extent of infection or metastasis, and condition of the patient(subject). It can generally be stated that a pharmaceutical compositioncomprising the T cells described herein may be administered at a dosageof 10⁴ to 10⁹ cells/kg body weight, preferably 10⁵ to 10⁶ cells/kg bodyweight, including all integer values within those ranges. T cellcompositions may also be administered multiple times at these dosages.The cells can be administered by using infusion techniques that arecommonly known in immunotherapy (see, e.g., Rosenberg et al, New Eng. J.of Med. 319: 1676, 1988). The optimal dosage and treatment regime for aparticular patient can readily be determined by one skilled in the artof medicine by monitoring the patient for signs of disease and adjustingthe treatment accordingly.

The administration of the subject compositions may be carried out in anyconvenient manner, including by aerosol inhalation, injection,ingestion, transfusion, implantation or transplantation. Thecompositions described herein may be administered to a patientsubcutaneously, intradermaliy, intratumorally, intranodally,intramedullary, intramuscularly, by intravenous (i.v.) injection, orintraperitoneally. In one embodiment, the T cell compositions of thepresent invention are administered to a patient by intradermal orsubcutaneous injection. In another embodiment, the T cell compositionsof the present invention are preferably administered by i.v. injection.The compositions of T cells may be injected directly into a tumor, lymphnode, or site of infection.

In certain embodiments of the present invention, cells activated andexpanded using the methods described herein, or other methods known inthe art where T cells are expanded to therapeutic levels, areadministered to a patient in conjunction with (e.g., before,simultaneously or following) any number of relevant treatmentmodalities, including but not limited to treatment with agents such asantiviral therapy, cidofovir and interleukin-2, Cytarabine (also knownas ARA-C) or natalizumab treatment for MS patients or efalizumabtreatment for psoriasis patients or other treatments for PML patients.In further embodiments, the T cells of the invention may be used incombination with chemotherapy, radiation, immunosuppressive agents, suchas cyclosporin, azathioprine, methotrexate, mycophenolate, and FK506,antibodies, or other immunotherapeutic agents. In a further embodiment,the cell compositions of the present invention are administered to apatient in conjunction with (e.g., before, simultaneously or following)bone marrow transplantation, or the use of chemotherapy agents such as,fludarabine, external-beam radiation therapy (XRT), cyclophosphamide.For example, in one embodiment, subjects may undergo standard treatmentwith high dose chemotherapy followed by peripheral blood stem celltransplantation. In certain embodiments, following the transplant,subjects receive an infusion of the expanded immune cells of the presentinvention. In an additional embodiment, expanded cells are administeredbefore or following surgery.

The dosage of the above treatments to be administered to a patient willvary with the precise nature of the condition being treated and therecipient of the treatment. The scaling of dosages for humanadministration can be performed according to art-accepted practices. Ingeneral, 1×10⁶ to 1×10 of the modified T cells of the invention (e.g.,CAR-T 20 cells) can be applied to patients by means of, for example,intravenous infusion each treatment or each course of treatment.

The Advantages of the Present Invention Include

(1) As for the chimeric antigen receptor of the present invention, theextracellular antigen binding domain thereof is a specific anti-CD20scFv. The CAR formed by binding the specific anti-CD20 scFv to aspecific hinge region and an intracellular domain shows a greatcapability of killing tumor cells with low cytotoxicity and low sideeffects.

(2) The chimeric antigen receptor provided by the invention can achievestable expression and membrane localization of CAR protein after T cellsis infected by lentivirus carrying CAR gene.

(3) The CAR-modified T cell of the present invention has a longersurvival time in vivo and strong anti-tumor efficacy. The optimized CARwith the IgG4 Hinge-CH2-CH3 linker region can avoid the binding of theFc receptor and the subsequent ADCC effect (antibody-dependentcytotoxicity).

Example 1 Construction of Lentiviral Expression Vector

The full-length DNA synthesis and cloning construction of codingplasmids were commissioned by Shanghai Boyi Biotechnology Co., Ltd.Different anti-CD20 scFv coding sequences were used in each plasmid. Thecloning vector was selected as pWPT lentiviral vector. The cloning siteswere BamH I and Sal 1 sites. The specific sequence structure is shown inFIG. 1. The amino acid and nucleotide sequences of each element are asdescribed above.

In the following examples, CAR-T20.13, CAR-T20.14, CAR-T20.16,CAR-T20.19, CAR-T20.20 with better effects are taken as examples.

Example 2 Preparation of CAR-T Cell

(1) After taking venous blood from healthy people, mononuclear cells(PBMCs) were isolated by density gradient centrifugation.

(2) On day 0, PBMCs were cultured in GT-T551 cell culture mediumcontaining 2% human albumin, and the final concentration of cells wasadjusted to 2×10⁶ cells/mL. The cells were seeded in a cell cultureflask previously coated with Retronectin (purchased from TAKARA) at afinal concentration of 10 μg/mL and CD3 monoclonal antibody (OKT3) at afinal concentration of 5 μg/mL. Recombinant human interleukin 2 (IL-2)was added to the culture medium at a final concentration of 1000 U/mL.The cells were cultured in an incubator with a saturated humidity and 5%CO₂ at 37° C.

(3) On day 2, fresh medium, concentrated and purified CAR20 lentivirussolution, protamine sulfate (12 μg/ml), and IL-2 (at a finalconcentration of 1000 U/mL) were added. After 12 hours of infection in a5% CO₂ incubator at 37° C., the culture medium was discarded, freshmedium was added, and cultivation was continued in a 5% CO₂ incubator at37° C.

(4) Starting from day 6, CART20 cells can be taken for the correspondingactivity assay.

In the present invention, the preparation process of CAR-modified T celltargeting CD20 antigen is improved, and GT-551 serum-free mediumsupplemented with 2% human albumin was selected to culture lymphocytesin vitro.

Example 3 Detection of the Integration Rate of the CAR Gene in the TCell Genome and the Expression Level of the Encoded Protein Thereof onthe Membrane Surface

0.5×10⁶ of CART-20 cell samples cultured on day 7 (FIG. 2A and FIG. 5A)and day 11 (FIG. 2B) in Example 2 were taken, respectively. Theexpression level of CAR20 protein on the surface of T cell membrane wasanalyzed by flow cytometry after Protein L staining. The results showedthat, except for CAR-T20.13, all of the CAR structures designed in thisstudy can detect the chimeric antigen receptor localization on the cellmembrane surface of the corresponding modified T cells using Protein L.

Example 4 Detection of the In Vitro Activation Ability of CAR-T20s

The deCAR-T20 cells cultured on day 6 in Example 2 were co-cultured withtarget cells. Then the up-regulated level of CD137 and the secretionlevel of IFNγ in the culture supernatant were examined. 1×10⁵ of CART-20cells (cultured on day 6) were cultured respectively with CD20-positiveRAM and RAMOS tumor cell lines, and CD20-negative MOLT-4 tumor cellline, or without tumor cells, in 200 μl GT-551 medium for 18 h in aratio of 1:1. Then the expression level of CD137 on the surface of Tcell membrane was detected by flow cytometry (FIG. 3A) and the secretionlevel of IFNγ in the culture supernatant was detected by ELISA (FIG.3B).

From the results in FIG. 3, we could concluded that the CAR based onObinutuzumab also achieved expression and membrane surface localizationin the corresponding modified cells, but the CAR structure based on theOfatumumab sequence showed better in vitro activation ability andspecificity targeting antigen when compared with the CAR constructedbased on Obinutuzumab and Rituximab.

Example 5 Detection of the CAR-T20s Cells Induced Early ApoptosisActivity of Tumor Cells

CART-20.13, CART-20.14 and CAR-T20.16 cells (cultured on day 11) fromExample 2 were co-cultured respectively with 1×10⁴ of CFSE-labeledCD20-negative (MOLT-4) or CD20-positive (RAJI, RAMOS) tumor cell linesin 200 μl GT-551 medium for 4 h. Then the cell pellet was collected bycentrifugation. The cells were washed twice with PBS and stained for 30min with Annexin V-APC dye in a ratio of 1:50 in 100 μl of dyeingsolution. After washing with PBSonce, the proportion of Annexin Vpositive cells in CFSE positive cells was analyzed on a flow cytometry.

The results in FIG. 4 show that the CAR structure based on theOfatumumab sequence shows better ability to induce early apoptosis ofCD20 target cells in vitro when compared with the CAR constructed basedon Obinutuzumab and Rituximab.

Example 6 Identification of the In Vitro Activation Ability of the ThirdGeneration Chimeric Antigen Receptor and the Chimeric Antigen Receptorwith Mutation in Hinge Region

(1) Under the condition that the transfection rate was basically equal(FIG. 5A), the CAR-T20s cells (prepared by the method of Example 2)cultured on the day 7 were cultured respectively with K562, K562 stabletransfected cells of CD19 single positive, CD20 single positive, CD19and CD20 double positive, and RAJI target cell (each taking 1×10⁵ cells)in 200 μl GT-551 medium for 18 h in a ratio of 1:1. Then theup-regulated level of CD137 (FIG. 5B) and the secretion level of IFNγ inthe culture supernatant (FIG. 5C) were detected.

(2) The results shown in FIG. 5 indicate that the in vitro activationability (CD137 and IFNg) of the chimeric antigen receptor CAR-T20.14 andCAR-T20.19 (having a mutation in the hinge region) is substantiallyequivalent, in the case of substantially identical infection efficiency.The third generation CAR structure CAR-T20.20 shows better in vitroactivation capacity (CD137 and IFNγ) than the second generationCAR-T20.14 and CAR-T20.19.

Example 7. Detection of the Ability of CAR-T20 Cells to Scavenge CD20Positive Cells In Vivo

(1) Raji-Luc cells expressing luciferase were injected into NCG mice(5×10⁵/mouse) through the tail vein. One week after the inoculation, thein vivo expansion of the tumor cells was observed by in vivo imaging andrecorded as Day 0. NT and CAR-T20.19 cells were injected into Day 0 mice(5×10⁶/mouse) through the tail vein. On Day0, Day7, Day14, Day21, theexpansion of tumor cells in mice was observed by in vivo imaging andanalyzed based on changes in fluorescence intensity and body weightchanges of mice.

(2) The results shown in FIG. 6 indicate that CAR-T20.19 can effectivelyinhibit the in vivo expansion of CD20-positive tumor cells.

The preferred embodiments of the present invention are described indetail above, but the present invention is not limited to the specificdetails in above embodiments. Various simple modifications can be madeto the technical solutions of the present invention within the scope ofthe technical idea of the present invention. These simple variants allfall within the protection scope of the present invention.

It should be further noted that the specific technical featuresdescribed in the above specific embodiments may be combined in anysuitable manner without contradiction. To avoid unnecessary repetition,the various possible combinations of the invention are not describedseparately.

In addition, any combination of various embodiments of the invention maybe made as long as it does not deviate from the idea of the invention,and it should be regarded as the disclosure of the invention.

The invention claimed is:
 1. A chimeric antigen receptor (CAR),comprising an amino acid sequence set forth in SEQ ID NO:
 5. 2. Animmune cell comprising a chimeric antigen receptor (CAR), wherein theCAR comprises an amino acid sequence set forth in SEQ ID NO:
 5. 3. Theimmune cell of claim 2, wherein the immune cell is a T cell.
 4. Apharmaceutical composition comprising an immune cell which comprises achimeric antigen receptor (CAR), wherein the CAR comprises an amino acidsequence set forth in SEQ ID NO: 5.